The overall objective of this proposal is to elucidate the molecular mechanisms by which elevated levels of glucose and fatty acids adversely affect the pancreatic -cell, a phenomenon referred to as glucolipodysfunction. The underlying hypothesis, based on our previous findings and preliminary data, proposes that early stages of glucolipodysfunction involve two complementary mechanisms: 1- Inhibition of expression of the enzyme Per Arnt Sim kinase (PASK) which results in diminished expression and activity of the transcription factors pancreatic-duodenal homeobox-1 (Pdx-1) and mammalian homologue of avian MafA/l-Maf (MafA), leading to decreased insulin expression via alterations of the histone code and a closed chromatin conformation; and 2- Nutrient-induced -cell proliferation driven by elevated circulating levels of fibroblast growth factor 21 (FGF21) activating FoxM1 signaling and leading to a dysfunctional -cell mass. In specific Aim 1 we will determine how inhibition of PASK expression impairs insulin gene expression in glucolipodysfunction. Our working hypothesis is that PASK phosphorylates, and thereby inactivates, glycogen synthase kinase (GSK) 3 which alleviates proteasomal degradation of Pdx-1 and MafA. Using rodent genetic models we propose to further delineate the functional relationship between PASK and GSK3 and its consequences on Pdx-1 and MafA expression and function under conditions of glucolipodysfunction. In specific Aim 2 we will characterize the modifications of the histone code and DNA methylation profile at the Pdx-1, MafA, and insulin promoters associated with glucolipodysfunction. Our working hypothesis is that Pdx-1 deficiency in glucolipodysfunction results in defective recruitment of the histone methyltransferase Set7/9 and alterations of the histone methylation profile at the insulin, Pdx-1, and MafA promoters. Using ex vivo and in vivo models we propose to identify the epigenetic modifications responsible for the initiation of glucolipodysfunction. In specific Aim 3 we will ascertain how insulin resistance induces -cell proliferation in glucolipodysfunction. Our working hypothesis is that insulin resistance in response to nutrient excess in 6-mo-old rats is associated with a rise in circulating factors, FGF21 being a likely candidate, which stimulate FoxM1- mediated -cell proliferation. Using in vivo models we propose to identify the mechanisms whereby insulin resistance promotes -cell growth under conditions of nutrient excess. We expect that the studies described in this application will reveal the molecular signature of glucolipodysfunction in the pancreatic -cell. We anticipate that these findings will serve as a basis to design novel therapeutic approaches to prevent the deterioration of -cell function in T2D. PUBLIC HEALTH RELEVANCE: Pancreatic -cell function inexorably declines during the course of type 2 diabetes mellitus. This deterioration is due, at least in part, to the metabolic perturbations associated with diabetes, chiefly chronic hyperglycemia and dyslipidemia, which themselves have detrimental effects on -cell function. By discovering the molecular mechanisms by which chronic excess of nutrients impair insulin production from the -cell we hope to identify potential therapeutic targets to curtail the degradation of glucose homeostasis in type 2 diabetes.